Process Paves Way for Zebrafish Knockout Bank

A new, more efficient technique for generating systematic zebrafish gene knockouts may soon provide the genomic research community with a comprehensive zebrafish gene knockout bank. In last week's online edition of the Proceedings of the National Academy of Sciences, researchers from the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health (NIH); the University of California, Los Angeles; and Peking University in Beijing, China, reported developing a technology to knockout zebrafish genes in a stable, targeted manner.

"Zebrafish have become a powerful model organism for studying vertebrate development," said senior author Shawn Burgess, Ph.D., investigator in the NHGRI Genome Technology Branch. "It is now possible to create a stable resource that contains mutations possibly in every known zebrafish gene."

Genomic researchers recognized relatively recently the attractiveness of zebrafish, Danio rerio, as a model organism. The small, transparent aquarium fish has many features that make it useful in studying the biology of vertebrates and in comparative genomics. It shares many of the same genes as humans, reproduces at a rapid rate, and its genes can be readily mutated. A project underway since 2001 and advancing toward completion is sequencing the 25 chromosome pairs of the zebrafish genome, a concurrent asset for building a zebrafish knockout resource.

The present research outlines the potential for stable, targeted inactivation of genes in zebrafish with an approach that uses retroviral mutagenesis and high-throughput mapping of retroviral integrations. The U.S.- China team proved the efficiency of a new technique that quickly identifies the zebrafish gene that has been knocked out by a random, retroviral insertion. To achieve this result, the researchers infected zebrafish embryos with a modified retrovirus and then mapped the locations of the retroviral insertions, or integrations. Retroviruses were originally designed for gene therapy because they can integrate their genetic material into the chromosomes of infected cells. That capability has now been repurposed to create knockout organisms.

Previous research has shown that retroviruses preferentially integrate their own genetic material near the beginning of the host cell's genes, potentially disrupting - or "knocking out" - the function of the normal gene. The retrovirus DNA sequences flag the location of the knocked-out gene, helping researchers identify it easily.

For the new process, a zebrafish embryo is first infected with retroviruses, creating thousands of different retroviral integrations. The infected zebrafish, called founders, are raised to adulthood and bred to produce the first filial generation of mutant zebrafish. Sperm from this generation is collected and frozen, and, at the same time, the genomic locations of the retroviral integrations are mapped. If one of the archived zebrafish has an integration in a gene that a researcher wants to study, the mutation can then be readily generated through in vitro fertilization of the frozen sperm sample.

The technique is an example of reverse genetics, where a researcher inactivates a specific gene of interest to determine its function by observing what happens in the absence of that gene. The use of retroviruses to knock out genes, as opposed to traditionally employed chemical mutagens or radiation, is a technique based on previous work by Dr. Burgess and others that reduced the time for gene identification from years to weeks.

"With classical genetics, many mutations are only seen if you ask the right question," explained Dr. Burgess. "We wanted a way to generate all of those invisible mutations and later have all of the genes knocked out so that later you could theoretically screen all genes for whatever biological measurement, or assay, you want to test."

Analysis of the technique demonstrated its success in three ways: improved retroviral injection techniques were on average three times more effective than the rate of previous methods; the gene mapping technique optimized consistency so that about 50 percent of the DNA sequences screened had unique integration sites, which makes the ultimate goal of a knockout for every gene in the zebrafish genome a more realistic possibility, both in terms of workload and cost; and the disruption of the gene expression - creating the knockout gene zebrafish - was proved functionally effective. Roughly 20 percent of the retroviral integrations into the embryo of the zebrafish caused a desired mutagenic outcome of greater than 70 percent reduction in gene activity, which is considered remarkable and is reason to put stock in this technique for generating the zebrafish knockout bank.

The new research also discovered the surprise result that viral integrations appear to disrupt gene expression levels only when they are in the gene's first intron, but this effect was very consistent, showing researchers a way to predict which retrovirus integrations are likely to cause a mutation and which ones are not.

Having demonstrated that knocking out all the genes in the zebrafish genome is a realistic possibility, the researchers have initiated collaboration between NIH and Peking University for a large-scale zebrafish gene knockout bank. At Peking University, the home of Dr. Burgess' collaborators, a very large zebrafish facility was recently built, which is more than 10 times larger than the size of Dr. Burgess' zebrafish facility at NIH. The Peking University facility has capacity to execute the high number of mutagenic events and fish breeding that, combined with the expertise at NIH, will enable the development of a zebrafish knockout bank.

"I could not do the next steps in my very small fish room here at NIH," said Dr. Burgess. "Peking University has been a valuable collaborator for this project and because of their very large facility we have the resources needed to do this project on a scale that would make it of significant impact. It is sort of a perfect union."

The zebrafish knockout bank envisioned by Dr. Burgess will entail a distribution capacity from a zebrafish stock center. NHGRI and Peking University are currently in discussions with the Zebrafish International Resource Center to see if distribution through that facility is possible. "NHGRI and Peking University established a cooperative agreement before the research collaboration got started so there was a clear institutional understanding that this will be a resource that's freely available to all scientists when we are done."

"It is a very large institutional commitment to zebrafish research to be sure," he added. "We foresee that if a researcher wants a particular gene mutation, they look it up on an Internet browser for the retroviral mutation in your gene of interest, order it and receive the mutant carrier fish in a matter of days."

The zebrafish knockouts would have enormous utility for research, particularly for studies of adult phenotypes, behavioral traits or cancer susceptibility. Probably 90 percent or more of the genes knocked out in the zebrafish genome will survive to adulthood, though traditionally most of the screening of zebrafish is done in the first five days of development because of the need for space to house so many fish.

Dr. Burgess envisions next undertaking the process to mutagenize every zebrafish gene using the techniques just published and eventually will use this resource to generate live knockout fish. "Conceivably, we can systematically test what would happen if we knocked out each and every gene in the zebrafish genome, and a 'living library' of all these gene knockouts could be housed in a large, but feasible, fish facility," he said. "On a good day mice have merely a fraction of that potential."